PROJECT SUMMARY/ABSTRACT Juvenile myelomonocytic leukemia (JMML) is a fatal childhood myeloproliferative neoplasm in which myeloid cells are overproduced and hematopoietic progenitors are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF). The only curative treatment is allogeneic bone marrow transplant, but even this rigorous therapy yields only a 50% relapse-free 5-year survival. A majority of patients have hyperactive Ras signaling, with the most common mutation being somatic gain-of-function (GOF) mutations in PTPN11, which encodes the protein tyrosine phosphatase Shp2. Previous work in the Chan lab has shown that the PI3K catalytic subunit p110? is needed for both Akt and Erk hyperactivation, and promotes GOF Shp2-induced GM- CSF hypersensitivity and hyperproliferation, contributing to the progression of JMML. Based on the significant role of p110? in promoting GOF Shp2-induced leukemia, we investigated potential tyrosine kinases that can cooperate with p110? to promote Akt and Erk activation and lead to hyperproliferation of myeloid cells. Bruton's Tyrosine Kinase (BTK) has been identified as a critical molecule in lymphoid malignancies and the BTK inhibitor, ibrutinib, was recently approved for use in patients with B cell leukemias. As BTK has been well- studied in the context of B cell receptor signaling, it is known that BTK is activated downstream of p110? and that active BTK phosphorylates B cell adaptor for PI3K (BCAP), allowing phospho-BCAP to bind to the regulatory p85? subunit and promote activation of PI3K. Based on what is known in B cells, we hypothesize that BTK and BCAP are critical players in the GOF Shp2-mediated hyperactivation of p110? signaling in JMML. To address this hypothesis, we propose three aims: (1) we will use murine cells expressing the GOF Shp2 mutation, E76K, with genetic knockout of BTK to perform proliferation and biochemical analysis in vitro, as well as examine the progression of myeloproliferative disease in p110? inhibitor-treated mice in vivo; (2) we will use cells from mice lacking expression of p85? along with p85? site-specific mutant constructs to explore the physical interaction between BCAP and p85?; and (3) we will test the efficacy of BTK (ACP-196) and p110? (ACP-319) inhibitors provided by AcertaPharma on Shp2E76K-expressing mice in vivo, as well as on primary JMML patient cells in vitro. These studies are timely and significant, as the AcertaPharma inhibitors are currently being tested in combination for safety and efficacy in chronic lymphocytic leukemia patients, and therefore have the potential to be a novel treatment in patients with JMML.